Zinc is the second most abundant microminerals in humans and is essential for life. Many diseases are associated with abnormal zinc metabolism. Zinc homeostasis in humans is primarily maintained by the adjustments of zinc absorption and excretion through zinc transporters. As one of two major zinc transporter families in mammals, Zrt- and Irt-like proteins (ZIP) facilitate zinc ions flux across membranes from extracellular spac or intracellular organelles into the cytoplasm. The fourteen ZIP proteins in humans share a conserved transmembrane domain (TMD) which forms the ion transport pathway, whereas the variable extracellular domains (ECD) are thought to play regulatory roles associated with the diverse functions of different ZIP proteins. The importance of the ECD is well documented in the study of ZIP4, a representative ZIP protein exclusively responsible for zinc absorption in the intestine. It has been established that missense mutations of ZIP4 cause a lethal genetic disorder, Acrodermatitis Enteropathica (AE), due to severe zinc deficiency. Notably, more than half of the AE-causing mutations of ZIP4 occur within the ECD. Previous biochemical and cell biological studies indicated that ZIP4-ECD plays crucial roles in zinc transport and ZIP4 protein regulation. However, the lack of high resolution three-dimensional structure of ZIP4-ECD prevents incisive structure-function studies. This is a major barrier to the understanding of the underlying mechanism of ZIP4-ECD function and why AE-causing mutations on ZIP4-ECD cause ZIP4 dysfunction and misregulation. In this work, we will focus on the structural and functional study of ZIP4-ECD and our central hypothesis is that ZIP4-ECD is a zinc binding domain and acts as both a zinc trap and a zinc sensor. In Specific Aim1, we will solve the crystal structure of a mammalian ZIP4-ECD. In Specific Aim2, we will characterize zinc binding and zinc binding induced conformational changes in ZIP4-ECD. In Specific Aim3, we will clarify the molecular mechanism of how AE-causing mutations on ZIP4-ECD impair the function and regulation of ZIP4, and establish the structure-function relationship of ZIP4-ECD. This work is significant because establishment of structure-function relationship of ZIP4-ECD will not only advance our knowledge about zinc transport through ZIP4 and the mechanism of ZIP4 regulation, but also promote the study of other ZIP proteins important in medicine. In terms of translational significance, ZIP4-ECD is an attractive drug targeting site and the high resolution structure of ZIP4-ECD will facilitate the design and development of ZIP4 inhibitors and activators. Several lines of evidences strongly suggested that ZIP4 inhibition is a promising strategy against cancer, particularly pancreatic cancer. In contrast, application of an allosteric agonist of ZIP4 may strengthen the effects of zinc supplement to the people at high risk of zinc deficiency, such as pregnant/lactating women and elders, by improving zinc absorption efficiency of ZIP4.